Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


The development of whole exome and whole genome sequencing technologies has allowed geneticists to gradually uncover a wide spectrum of rare, deleterious mutations with large effect sizes, which are responsible for Mendelian diseases. Roughly 70% of human genes are single copy and have orthologs across the vertebrate lineage, suggesting that they are under purifying selection. This suggests that at least 10,000 genes will have Mendelian phenotypes when mutated but have yet to be discovered1. Exhaustive research efforts have been directed at finely categorizing the coding, and more recently noncoding, genome. The actual number of large effect noncoding mutations, and of particular importance given their scarcity, mutations in functional regulatory regions have yet to be determined. Ideal prototypes to study these variants are recessive Mendelian diseases that are relatively common in the population, caused only by biallelic mutations in one gene, have virtually 100% penetrance with consistent phenotypic expressivity with recessive genotypes, and few, if any, phenotypic features in subjects with heterozygous mutant alleles. To date approximately 10 genes (SLC12A3, SLC12A1, KCNJ1, CLCNKB, BSND, MAGED2, KCNJ10, CLDN10, CLDN16, CLDN19) have been linked to recessive salt wasting disorders. Gitelman syndrome (GS) is a recessive Mendelian kidney disorder caused by deleterious mutations in SLC12A3 and has an incidence of approximately 1 in 40,0002. Clinical hallmarks of GS include hypotension, hypokalemia, hypomagnesemia, metabolic alkalosis, and hypomagnesemia. To further decipher the full contribution of regulatory mutations to Mendelian disorders, blood samples from a cohort of 387 patients referred to us by nephrologists for suspected salt-wasting disorders were subjected to whole-exome analysis and subsequent whole-genome sequencing analysis on a subset of these patients. Whole exome sequencing analysis on the 387 electrolyte-wasting subjects identified 508 deleterious coding mutations in the 10 genes linked to Mendelian electrolyte-wasting in 254 subjects. A total of 17 patients with Gitelman syndrome harbored only monoallelic, deleterious mutations in SLC12A3 and were subjected to whole genome sequencing to identify any putative large effect noncoding variants in SLC12A3. Whole genome sequencing analyses identified a total of 14 mutations outside of the coding regions in SLC12A3. These 14 mutations include 7 large deletions of two or more exons, 2 deep intronic mutations that introduce cryptic donor splice sites, 1 in-frame 6 base pair cryptic insertion at the intron-exon boundary, 3 mutations at noncanonical splice regions that disrupt conventional splicing, and 1 mutation within a putative SLC12A3 cis-regulatory site. A luciferase reporter assay revealed a drastic decrease in reporter activity for the regulatory variant and indicates this variant introduces a novel repressor site in intron 1 of SLC12A3. Collectively, whole exome and whole genome analyses identified biallelic mutations in 70% of all subjects with suspected electrolyte-wasting that likely represent causal genetic lesions, with regulatory variants only comprising ~0.2% of deleterious alleles.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Monday, December 25, 2023

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